The present invention relates to methods of generating tissue using scaffold matrices derived from micro-organs and stem cells of embryonic or adult origin. The present invention further relates to novel approaches for inducing differentiation of adult or embryonic stem cells for isolating adult stem cells and a method of continuously generating stem cells by implantation of micro-organs as sources of stem cells.
Stem Cells (SCs)
Until very recently it was believed that only certain embryonic cells retain the capacity or competence of being able to differentiate into most, if not all, of the cell types that constitute the vertebrate body (pluripotent stem cells). With the advent of bone marrow transplantation and other remarkable experiments it is now clear that also in the vertebrate adult including man there exists in tissues like bone marrow, adipose tissue and brain, true stem cells with the capacity or competence to differentiate into many other cell types, in a process generally referred to as transdifferentiation, if provided with the appropriate stimuli (Bjorson et al 1999, Mezey et al 2000, Orlic et al 2001, Kocher et al 200). Prior to the publication of these remarkable experiments, it was believed that there existed stem cells in some adult tissues like gut and skin, but that the differentiation potential of these stem cells in adult tissues is limited to cell lineages present in the organ from which they were derived.
Recent studies have shown that certain stem cells in adult tissue express more of a “pluripotent character” than previously thought. Bone marrow (BM) cells were shown to differentiate into various cell types, including liver. Neural stem cells were recently shown to be capable of repopulating the hematopoietic system, producing blood cells and integrating and differentiating into various other tissue types. Other examples are also known. In these examples “real” stem cells (SCs) were used, in the sense that they can give rise to cells from organs different than the organ of origin. In most of these examples, the SC derived from any of the three germ layers, the ectoderm, the mesoderm or the endoderm have been shown to differentiate into cells whose phenotype corresponds to a different germ layer, e.g., ectoderm into mesoderm, etc.
Clearly then, SCs exist both in early embryos and in adult tissues. While SCs are now known to have a competence to differentiate along various lineages, the following goals still await further developments (i) to find protocols with which to induce SCs differentiation along a differentiation lineage of choice; and (ii) once differentiated, to incorporate SCs into organized complex three-dimensional structures or tissues that constitutes the vertebrate body.
The present invention offers general and applicable solutions to these two goals.
Embryonic Stem Cells
Embryonic stem (ES) cells (ESCs) have commonly been derived from pre-implantation embryos. When cultured under proper conditions (fibroblast feeder layer or the addition of Leukemia inhibitory factor (LIF)) ESCs maintain both the ability to multiply in culture and their totipotential capacity (Keller, 1995). In this aspects as well as in their gene expression pattern, ESCs resemble the inner cell mass (ICM) of the developing embryo (O'Shea, 1999). The following examples provide insight with respect to the differentiation potential of ESCs:
(i) When injected into the ICM of a blastocyst, ESCs integrate into the ICM and populate all cell lineages including the germ line.
(ii) When injected subcutaneously into syngeneic mice, teratocarcinoma tumors, which contain cells of different embryonic origins develop (Rudnicki and McBurney, 1987).
(iii) When cultured in vitro, ESCs aggregate and generate colonies known as embryoid bodies (EB) (Keller, 1995). Overexpression of hepatocyte nuclear factor 3 (HNF3) was shown to promote ESCs' differentiation into endodermal lineage in vitro (Levinson-Dushnik and Benvenisty, 1997).
Adult Stem Cells
It was originally thought that only ESCs are totipotent. The differentiation potential of SCs in adult tissue was thought to be limited to cell lineages present in the organ from which they were derived. Recent studies have shown that certain stem cells in adult tissue express more of a “pluripotent character” than previously thought. For instance, bone marrow (BM) cells were shown to differentiate into various cell types, including liver cells. Hemotopoetic SCs were recently shown to be capable of differentiating into neurons (Mezey et al 2000; Recently Kocher et al. (2001) have reported neovascularization of ischemic myocardium of athymic nude rats by cytokine-mobilized human bone-marrow-derived stem cells (Kocher et al 2001). In such experiments, as well as in the experiments reported by Orlic et al. (2001), homing of the stem cells was attributed to the acute injury inflicted by the experimental procedure.
Micro Organs (MOs)
Micro organs are organ portions of unique characters. On the one hand, MOs are of sufficient size so as to preserve the micro-architecture of the tissue or organ from which they were derived. On the other hand, they are of dimensions which allow efficient nutrients and wastes exchange by diffusion with the growth medium in which they are kept. As such, MOs retain viability in culture for as long as 45 days or more, and they were shown to transcribe tissue specific genes throughout this time period. Furthermore, since cell interactions are kept and different cell types of the MO are cultured together, MOs can be cultured in serum-free media. The development of the MOs technology which is further described in U.S. Pat. No. 5,888,720, and PCT Applications No. IL00/00365, IL00/00424 and US98/00594, all of which are incorporated herein by reference, was following the observation that every tissue in the body of a multi-cellular organism having a blood system is composed of tissue units, each such unit is composed of numerous cell types, each of which is positioned not more than 225–300 μm away from a nearest blood vessel, which distance is, in effect, dictated by diffusion rates of nutrients and wastes. This observation was translated, as described in the above Patent and Applications, into the MOs technology, according to which an MO is prepared from a tissue so as to include all the cell types of a tissue unit, each of which is positioned not more than 225–300 μm away from a growth medium in which the MO is placed, which distance allows efficient diffusion rates of nutrients and wastes, so as to sustain MO viability in minimal (serum free) medium for long time periods.
There is thus a highly recognized need for, and it would be highly advantageous to have methods of generating adult stem cells and methods of utilizing adult and embryonic stem cells for generating differentiated and functional tissues.